The present invention relates to a confocal microscopy apparatus and method and in particular to confocal microscopy apparatus that enables real-time imaging to be performed.
Confocal microscope systems were originally designed to generate a substantially pure confocal image by scanning a diffraction limited spot of light across the object being imaged. Light reflected from separate scanned points of the object was sequentially received by a series of photodetectors to gradually build up a 2-D confocal image of the object. Such confocal systems have the disadvantage that a very bright light source such as a laser is necessary and real-time imaging could not easily be performed because of the time required to scan over the surface of the object.
More recent developments have focused on ways of increasing the speed with which confocal images can be generated to enable real-time imaging. In WO97/31282 a confocal microscope is described in which a mask is used to generate a combined confocal and non-confocal image. The non-confocal image is subsequently subtracted from the combined image to provide a substantially pure confocal image. The mask in WO97/31282 consists of different, separated regions with a first region being substantially transparent to incident light and unpatterned and a second region being patterned, for example with an irregular array of spots that are opaque to incident light. Light that passes through the first region illuminates the object to generate a conventional image of the object. On the other hand, light that passes through the second region produces a pattern of the mask on the surface of the object that in turn generates a combined confocal and conventional image of the object. The mask is spun so that the first and second regions of the mask alternately transmit the illumination to the object and so generate alternate images of the object. The alternate images are then subtracted from one another so that the conventional image is removed from the combined image to leave only the confocal image.
An alternative theoretical design of a confocal microscope is described in a paper that appeared in the Journal of Microscopy, Vol. 189, Pt 3, March 1998 entitled xe2x80x9cTheory of confocal fluorescence imaging in the programmable array microscope (PAM)xe2x80x9d, Verveer et al. The programmable array microscope described in this paper consists of a spatial light modulator (SLM) in the form of a digital micromirror device (DMD). The individual pixels of the SLM are programmed to generate an arbitrary pattern of conjugate illumination and detection apertures and two separate cameras are used, one positioned to view active (xe2x80x98onxe2x80x99) micromirrors of the SLM and to receive the confocal reflected light from the object and one to view inactive (xe2x80x98offxe2x80x99) micromirrors and to receive a nonconjugate image of the object. However, significant difficulties have been encountered in developing the theoretical design to a working model and the design has never been made to work convincingly.
The present invention seeks to provide improved confocal microscopy apparatus that enables real-time imaging of an object and generates images containing less noise than is generally possible with current systems.
The present invention provides confocal microscopy apparatus comprising means for directing light to a specimen; at least one mask for encoding light incident on the specimen and for decoding light from the specimen to thereby generate a first image of the specimen consisting of a confocal image superimposed on a non-confocal image; and detection means for detecting said first image characterised in that there is further provided encoding means for encoding light to or from the specimen in reverse with respect to the encoding of the at least one mask to thereby generate a second image of the specimen consisting of a non-confocal image from which a confocal image has been removed.
Ideally, analysing means are provided for generating a confocal image of the specimen using the first and second images.
In a preferred embodiment a single mask is provided that encodes light incident on the specimen and decodes light from the specimen. The encoding means may consist of reflective patterning on one surface of the mask.
The microscopy apparatus may include one or more light sources. In particular, two light sources may be provided to illuminate each side of the mask respectively. Furthermore, the detection means may detect both the first and second images.
Alternatively, a second detection means may be provided for detecting the second image, the first and second detection means being located on opposite sides of the mask. The second detection means may be located to receive light reflected from said reflective patterning.
The mask may be patterned with equidistant light transmissive stripes and light barring stripes or with a random patterning. Also, the mask may be mounted for rotation about the optical axis of the apparatus.
In an alternative aspect the present invention provides a confocal microscopy method comprising providing illumination from a first light source; encoding light incident on a specimen and decoding light from the specimen by means of one or more masks; generating a first image of the specimen consisting of a confocal image superimposed on a non-confocal image; and detecting said first image characterised by encoding light to or from the specimen in reverse with respect to the encoding of the one or more mask and generating a second image of the specimen consisting of a non-confocal image from which a confocal image has been removed.
Ideally, the method further comprises generating a confocal image from said first and second images. Preferably, the second image is subtracted from the first image to extract the confocal image.
In a preferred embodiment a single mask is used to encode light to the specimen and to decode light from the specimen. Also, the second image may be generated by reflecting light off reflective patterning on one surface of said single mask. The mask may be rotatable.